Group A Streptococcus (GAS) is a major human pathogen producing invasive infections including necrotizing fasciitis (NF). The virulence factors responsible for bacterial spread and tissue injury in GAS NF are poorly understood. GAS are recognized phenotypically by a zone of beta-hemolysis produced largely by the cytolytic toxin streptolysin S (SLS). Our laboratory has led a collaboration that elucidated the genetic basis for SLS production. The 9-gene sag operon is both necessary for GAS SLS production and sufficient to confer SLS activity to the nonpathogenic heterologous species Lactococcus lactis. Sequence features and homologies strongly suggest SLS belongs to the bacteriocin class of toxins, with sagA encoding the toxin precursor (pre-SLS) and downstream genes (sagB-I) encoding chemical modification, processing and export functions. Target mutagenesis of each gene in the sag operon results in an SLS-negative phenotype. In vivo testing of SLS-negative sag knockout mutants in a mouse model of GAS NF showed that SLS is required for virulence. SLS-negative mutants failed to produce the necrotic ulcer, diffuse neutrophilic infiltrate, and widespread dermal and fascial tissue injury observed with the parent GAS strains. Our discovery and genetic analysis of the sag locus for SLS production has generated powerful information and reagents to study the molecular basis, biologic activities, and virulence properties of this GAS exotoxin. We hypothesize that each gene in the sag operon is required for proper expression of SLS, and that the SagA precursor is chemically altered, exported and processed to yield a mature protein with modified amino acids and structural features of a bacteriocin. We further hypothesize that GAS is a multifunctional toxin with cytotoxic and proinflammatory activities on host cells. Finally, we hypothesize that SLS plays an important role in the pathogenesis of GAS NF, through direct cytotoxicity, stimulation of neutrophil inflammation and interference with phagocytosis, perhaps acting synergistically with other GAS factors such as M-protein and SPE-B. These hypotheses will be tested by molecular genetic studies, attempts protein purification and antibody development, and the use of targeted SLS mutants in in vitro assays of phagocytic function and our in vivo mouse model of GAS NF.